Rapid Identification of Quox-1 Homeodomain DNA-Binding Sequence Using SAAB

Quox-1 is the only gene in the hox family whose expression occurs throughout the development of the central nervous system. Using the Quox-1 homeodomain produced in a bacterial expression system, we were able to identify DNA-binding targets of the Quox-1 protein from a library of randomly generated oligonucleotides by the selection and amplification binding (SAAB) technique. The results indicated that the Quox-1 protein recognizes a new consensus sequence, 5'-CAATC-3', which has not been reported for any other Hox family homeoprotein. In addition, electromobility shift assay further confirmed that the Quox-1 homeoprotein preferentially binds to the 5'-CAATC-3' sequence, but not to the binding sites for other Hox class homeoprotein (TAAT) or NKX class homeoprotein (CAAG). Based on mutation analyses of the DNA sequences, we found that the 5'-CAATC-3' core sequences are required for high affinity binding by the Quox-1 protein. Furthermore, mutation analyses of the Quox-1 homeodomain showed that one of the major determinants participating in recognition of a minor groove is the Gln6 and Thr7 in the N-terminal arm of the homeodomain.     

Nucleotides flanking a conserved TAAT core dictate the DNA binding specificity of three murine homeodomain proteins.

Murine homeobox genes play a fundamental role in directing embryogenesis by controlling gene expression during development. The homeobox encodes a DNA binding domain (the homeodomain) which presumably mediates interactions of homeodomain proteins with specific DNA sites in the control regions of target genes. However, the bases for these selective DNA-protein interactions are not well defined. In this report, we have characterized the DNA binding specificities of three murine homeodomain proteins, Hox 7.1, Hox 1.5, and En-1. We have identified optimal DNA binding sites for each of these proteins by using a random oligonucleotide selection strategy. Comparison of the sequences of the selected binding sites predicted a common consensus site that contained the motif (C/G)TAATTG. The TAAT core was essential for DNA binding activity, and the nucleotides flanking this core directed binding specificity. Whereas variations in the nucleotides flanking the 5' side of the TAAT core produced modest alterations in binding activity for all three proteins, perturbations of the nucleotides directly 3' of the core distinguished the binding specificity of Hox 1.5 from those of Hox 7.1 and En-1. These differences in binding activity reflected differences in the dissociation rates rather than the equilibrium constants of the protein-DNA complexes. Differences in DNA binding specificities observed in vitro may contribute to selective interactions of homeodomain proteins with potential binding sites in the control regions of target genes.     

Structural determinants within Pbx1 that mediate cooperative DNA binding with pentapeptide-containing Hox proteins: proposal for a model of a Pbx1-Hox-DNA complex.

Genetic studies have identified a family of divergent homeodomain proteins, including the human protooncoprotein Pbx1 and its drosophila homolog extradenticle (Exd), which function as cofactors with a subset of Hox and HOM-C proteins, and are essential for specific target gene expression. Pbx1/Exd binds DNA elements cooperatively with a large subset of Hox/HOM-C proteins containing a conserved pentapeptide motif, usually YPWMR, located just N terminally to their homeodomains. The pentapeptide is essential for cooperative DNA binding with Pbx1. In this study, we identify structural determinants of Pbx1 that are required for cooperative DNA binding with the pentapeptide-containing Hox protein HoxA5. We demonstrate that the homeodomain of Pbx1 contains a surface that binds the pentapeptide motif and that the Pbx1 homeodomain is sufficient for cooperative DNA binding with a Hox protein. A sequence immediately C terminal to the Pbx1 homeodomain, which is highly conserved in Pbx2 and Pbx3 and predicted to form an alpha-helix, enhances monomeric DNA binding by Pbx1 and also contributes to maximal cooperativity with Hox proteins. Binding studies with chimeric HoxA5-Pbx1 fusion proteins suggest that the homeodomains of Pbx1 and HoxA5 are docked on the representative element, TTGATTGAT, in tandem, with Pbx1 recognizing the 5' TTGAT core motif and the Hox protein recognizing the 3' TGAT core. The proposed binding orientation permits Hox proteins to exhibit further binding specificity on the basis of the identity of the four residues 3' to their core binding motif.     

Two highly related homeodomain proteins, Nkx5-1 and Nkx5-2, display different DNA binding specificities.

The mouse Nkx5-1 and Nkx5-2 genes are related to NK genes in Drosophila and encode proteins with very similar homeodomains. In higher vertebrates Nkx5 genes are specifically expressed in the inner ear. Inactivation of the mouse Nkx5-1 gene by homologous recombination revealed a critical role for the formation of vestibular inner ear structures. Here, we investigated biochemical properties of the proteins encoded by the Nkx5 genes. A similar consensus binding sequence was isolated for both Nkx5 proteins using binding site selection. This sequence is related to target sequences previously identified for other Nkx proteins and contains the conserved homeodomain binding core TAAT. An additional, novel and unrelated high affinity binding sequence could be identified for the Nkx5-2 protein.     

Unusual gene order and organization of the sea urchin hox cluster

While the highly consistent gene order and axial colinear patterns of expression seem to be a feature of vertebrate hox gene clusters, this pattern may be less well conserved across the rest of the bilaterians. We report the first deuterostome instance of an intact hox cluster with a unique gene order where the paralog groups are not expressed in a sequential manner. The finished sequence from BAC clones from the genome of the sea urchin, Strongylocentrotus purpuratus, reveals a gene order wherein the anterior genes (Hox1, Hox2 and Hox3) lie nearest the posterior genes in the cluster such that the most 3' gene is Hox5. (The gene order is 5'-Hox1, 2, 3, 11/13c, 11/13b, 11/13a, 9/10, 8, 7, 6, 5-3'.) The finished sequence result is corroborated by restriction mapping evidence and BAC-end scaffold analyses. Comparisons with a putative ancestral deuterostome Hox gene cluster suggest that the rearrangements leading to the sea urchin gene order were many and complex.     

Novel interactions between vertebrate Hox genes

Understanding why metazoan Hox/HOM-C genes are expressed in spatiotemporal sequences showing colinearity with their genomic sequence is a central challenge in developmental biology. Here, we studied the consequences of ectopically expressing Hox genes to investigate whether Hox-Hox interactions might help to order gene expression during very early vertebrate embryogenesis. Our study revealed conserved autoregulatory loops for the Hox4 and Hox7 paralogue groups, detected following ectopic expression Hoxb-4 or HOXD4, and Hoxa-7, respectively. We also detected specific induction of 5' posterior Hox genes; Hoxb-5 to Hoxb-9, following ectopic expression of Hoxb-4/HOXD4; Hoxb-8 and Hoxb-9 following ectopic expression of Hoxa-7. Additionally, we observed specific repression of 3' anterior genes, following ectopic expression of Hox4 and Hox7 paralogues. We found that induction of Hoxb-4 and Hoxb-5 by Hoxb-4 can be direct, whereas induction of Hoxb-7 is indirect, suggesting the possibility of an activating cascade. Finally, we found that activation of Hoxb-4 itself and of posterior Hox genes by Hoxb-4 can be both non-cell-autonomous, as well as direct. We believe that our findings could be important for understanding how a highly ordered Hox expression sequence is set up in the early vertebrate embryo.     

Homeodomain binding sites in the 5' flanking region of the Bombyx mori silk fibroin light-chain gene

Multiple A + T-rich stretches in the 5' flanking region of the Bombyx mori fibroin light-chain gene have been shown to bind two Drosophila homeodomain proteins, EVE (even-skipped) and ZEN (zerknüllt), with high affinities. Some of these sites fall into a class that has the established consensus sequence of the binding sites (TCAATTAAAT) for a diverse group of Drosophila homeodomain proteins, while others are quite heterogenous except that they all possess a core TAAT motif. Since clusters of homeodomain binding sites can also be found in the promoters of other silk protein genes, the fibroin gene and the sericin-1 gene, these observations suggest a possible involvement of some homeobox genes in the regulation of a group of silk protein genes.     

Location of the 11 bp exon in the chicken pro alpha 2(I) collagen gene.

During the fine structural analysis of the 5' end of the 38 kb chicken pro alpha 2(I) collagen gene, we failed to locate an exon, only 11 bp in size, which had been predicted from the DNA sequence analysis of a cDNA clone complementary to the 5' end of the pro alpha 2(I) collagen mRNA (1). We know report the location of this 11 bp exon, exon 2, at the 5' end of a 180 bp Pst I fragment, 1900 bp 3' to exon 1 and 600 bp 5' to exon 3. Its sequence, ATGTGAGTGAG, is highly unusual in that it contains two overlapping consensus donor splice sequences. Moreover, it is flanked by two overlapping donor splice sequences but only one of the four splice sequences is actually spliced (1). The first half of intron 1 also has an unusual sequence: it is 68% GC, contains 88 CpG dinucleotides and 11 Hpa II sites. The second half is more like other intron sequences in the collagen gene with a GC content of 41%, 19 CpG, and no Hpa II sites. However it contains two sequences with 7 and 9 bp homology to the 14 bp SV40 enhancer core sequence. It is suggested that some part of intron 1 may be involved in regulation.     

Sequence specificity of DNA cleavage by bis(1,10-phenanthroline)copper(I)

The bis(1,10-phenanthroline)copper(I) complex is a relatively simple molecule previously shown to cause DNA cleavage with a strong preference for gene control regions such as the Pribnow box. Sequence level mapping of sites of [(Phen)2CuI]+ cleavage in greater than 2000 bases in histone genes and the plasmid pUC9 showed that the specificity for control regions is related to a predominant preference for minor groove binding at TAT triplets, which were cleaved most strongly at the adenosine sugar ring. The related sequences TGT, TAAT, TAGPy, and CAGT (Py = pyrimidine) were moderately preferred, while CAT and TAC triplets, PyPuPuPu quartets, PuPuPuPy quartets, and CG-rich PyPuPuPy quartets were cleaved with low to average frequency. Polypurine and polypyrimidine sequences were cleaved with low frequency. The sequence preferences of [(Phen)2CuI]+ can be ascribed predominantly to (i) a requirement for binding in the minor groove at a pyrimidine 3'----5' step and (ii) stereoelectronic effects of the 2-amino group of guanine in the minor groove, which inhibit binding. Although the reagent appears primarily to recognize sequence features at the triplet or quartet level, lower than expected cleavage was observed for two TAT sequences adjacent to several other preferred sequences and higher than expected cleavage was observed at CAAGC sequences, suggesting that longer range sequence-dependent DNA conformational effects influence specificity in certain cases.     

Nucleotide sequences of Caenorhabditis elegans core histone genes. Genes for different histone classes share common flanking sequence elements

We have determined the nucleotide sequence of core histone genes and flanking regions from two of approximately 11 different genomic histone clusters of the nematode Caenorhabditis elegans. Four histone genes from one cluster (H3, H4, H2B, H2A) and two histone genes from another (H4 and H2A) were analyzed. The predicted amino acid sequences of the two H4 and H2A proteins from the two clusters are identical, whereas the nucleotide sequences of the genes have diverged 9% (H2A) and 12% (H4). Flanking sequences, which are mostly not similar, were compared to identify putative regulatory elements. A conserved sequence of 34 base-pairs is present 19 to 42 nucleotides 3' of the termination codon of all the genes. Within the conserved sequence is a 16-base dyad sequence homologous to the one typically found at the 3' end of histone genes from higher eukaryotes. The C. elegans core histone genes are organized as divergently transcribed pairs of H3-H4 and H2A-H2B and contain 5' conserved sequence elements in the shared spacer regions. One of the sequence elements, 5' CTCCNCCTNCCCACCNCANA 3', is located immediately upstream from the canonical TATA homology of each gene. Another sequence element, 5' CTGCGGGGACACATNT 3', is present in the spacer of each heterotypic pair. These two 5' conserved sequences are not present in the promoter region of histone genes from other organisms, where 5' conserved sequences are usually different for each histone class. They are also not found in non-histone genes of C. elegans. These putative regulatory sequences of C. elegans core histone genes are similar to the regulatory elements of both higher and lower eukaryotes. The coding regions of the genes and the 3' regulatory sequences are similar to those of higher eukaryotes, whereas the presence of common 5' sequence elements upstream from genes of different histone classes is similar to histone promoter elements in yeast.     

The optimal binding sequence of the Hox11 protein contains a predicted recognition core motif.

HOX11 is a homeobox-containing oncogene of specific T-cell leukemias. We determined the DNA binding specificity of the Hox11 protein by using a novel technique of random oligonucleotide selection developed in this study. The optimal Hox11 binding sequence, GGCGGTAAGTGG, contained a core TAAGTG motif that is consistent with a prediction based on the residues at specific positions that potentially make DNA base contacts and models of homeodomain-DNA interaction proposed from studies with other homeodomains. The specific interaction between Hox11 and the selected optimal binding sequence was further confirmed by band-shift and DNA competition assays. Given that the Hox11 homeodomain shares low homology with other well studied homeodomains, the presence of a predictable recognition core motif in its optimal binding sequence supports the notion that different homeodomains interact with DNA in a similar manner, through highly conserved residues at specific positions that allow contact with DNA.     

Phosphorylation influences the binding of the yeast RAP1 protein to the upstream activating sequence of the PGK gene.

Yeast repressor activator protein 1 (RAP1) binds in vitro to specific DNA sequences that are found in diverse genetic elements. Expression of the yeast phosphoglycerate kinase gene (PGK) requires the binding of RAP1 to the activator core sequence within the upstream activating sequence (UAS) of PGK. A DNA fragment Z+ which contains the activator core sequence of the PGK(UAS) has been shown to bind RAP1. Here we report that phosphatase treatment of RAP1 affected its binding to the PGK(UAS) but that this depended on the nature of the sequence flanking the 5' end of the activator core sequence. When the sequence flanking the 5' end of the activator core sequence was different from the PGK RAP1-binding site, phosphatase treatment of RAP1 decreased its binding to the DNA. When the 5' end of the binding site was a match to the PGK RAP1-binding site dephosphorylation of RAP1 increased RAP1 binding to the DNA. These observations were reproduced when the minimal functional DNA-binding domain of the RAP1 protein was used, implicating a phosphorylation-dependent binding of RAP1. This is the first evidence for phosphorylation-dependent binding of RAP1.     

Partial characterization of bovine elastin gene; comparison with the gene for human elastin

A 14 kilobase (kb) genomic clone of the gene for bovine elastin, containing exons 1 and 2, has been characterized. This clone extends about 6.5 kb in the 5' direction from the initiation codon and 978 nucleotides in the 3' direction from exon 2. The size of the first intron is about 6.4 kb. The sequence immediately 5' to the initiation codon is highly conserved between the genes for bovine and human elastins and contains a TATA box consensus sequence (ATAAA), CAAT, and Sp1 binding sites. Several putative AP-2 binding sites are also present. Comparative analysis of the sequences flanking the first exon in the genes for bovine and human elastins identified conserved sequences that may be regulatory control elements. A putative enhancer core sequence is present in the first intron of the genes for bovine and human elastins.     

Cdx protein interaction with Hoxa5 regulatory sequences contributes to Hoxa5 regional expression along the axial skeleton

Hox gene functions are intimately linked to correct developmental expression of the genes. The identification of cis-acting regulatory sequences and their associated trans-acting factors constitutes a key step in deciphering the mechanisms underlying the correct positioning of the functional domain of Hox genes along the anterior-posterior axis. We have identified DNA elements driving Hoxa5 regionalized expression in mice, using the 2.1-kb mesodermal enhancer (MES) localized in Hoxa5 3' flanking sequences as a starting point. The MES sequence comprises regulatory elements targeting Hoxa5 expression in the limbs, the urogenital and gastrointestinal tracts, and the cervical-upper thoracic region of the prevertebral column. A 164-bp DNA fragment within the MES caudally restricts Hoxa5 expression at the level of prevertebra 10, corresponding to the posterior limit of its functional domain. Cdx proteins directly bind to this element in vitro via two conserved sites. Preventing Cdx binding by mutating the sites causes caudal expansion of the transgene expression domain. Of all three murine Cdx proteins that bind this element in vitro, Cdx4 has emerged as a potential regional posterior repressor of Hoxa5 expression. The restrictive control provided by Cdx interactions with Hoxa5 regulatory sequences may be one of the critical events in cervicothoracic axial specification.